Chromosomal rearrangements played an important role in the speciation of rice rats of genus Cerradomys (Rodentia, Sigmodontinae, Oryzomyini)

Rodents of the genus Cerradomys belong to tribe Oryzomyini, one of the most diverse and speciose groups in Sigmodontinae (Rodentia, Cricetidae). The speciation process in Cerradomys is associated with chromosomal rearrangements and biogeographic dynamics in South America during the Pleistocene era. As the morphological, molecular and karyotypic aspects of Myomorpha rodents do not evolve at the same rate, we strategically employed karyotypic characters for the construction of chromosomal phylogeny to investigate whether phylogenetic relationships using chromosomal data corroborate the radiation of Cerradomys taxa recovered by molecular phylogeny. Comparative chromosome painting using Hylaeamys megacephalus (HME) whole chromosome probes in C. langguthi (CLA), Cerradomys scotii (CSC), C. subflavus (CSU) and C. vivoi (CVI) shows that karyotypic variability is due to 16 fusion events, 2 fission events, 10 pericentric inversions and 1 centromeric repositioning, plus amplification of constitutive heterochromatin in the short arms of the X chromosomes of CSC and CLA. The chromosomal phylogeny obtained by Maximum Parsimony analysis retrieved Cerradomys as a monophyletic group with 97% support (bootstrap), with CSC as the sister to the other species, followed by a ramification into two clades (69% of branch support), the first comprising CLA and the other branch including CVI and CSU. We integrated the chromosome painting analysis of Eumuroida rodents investigated by HME and Mus musculus (MMU) probes and identified several syntenic blocks shared among representatives of Cricetidae and Muridae. The Cerradomys genus underwent an extensive karyotypic evolutionary process, with multiple rearrangements that shaped extant karyotypes. The chromosomal phylogeny corroborates the phylogenetic relationships proposed by molecular analysis and indicates that karyotypic diversity is associated with species radiation. Three syntenic blocks were identified as part of the ancestral Eumuroida karyotype (AEK): MMU 7/19 (AEK 1), MMU 14 (AEK 10) and MMU 12 (AEK 11). Besides, MMU 5/10 (HME 18/2/24) and MMU 8/13 (HME 22/5/11) should be considered as signatures for Cricetidae, while MMU 5/9/14, 5/7/19, 5 and 8/17 for Sigmodontinae.


BI
The employment of whole chromosome probes to study karyotype diversity provides precise information about the chromosomal rearrangements involved in intra-and interspecific differences among many species 1,2 , and identifies chromosomal traits that can be used as phylogenetic markers [3][4][5][6] .Indeed, the use of human whole chromosome probes (HSA) confirms the monophyly of Xenarthra (sloths, armadillos, and anteaters) 7 and Afrotheria 8 .The detection of the trait HSA 2q/21 elucidates the ambiguous position of Dermoptera within Supraprimates and shows that Scandentia and Dermoptera are more phylogenetically related to each other than to Primates 9 ; and in Phyllostomidae bats, the monophyly of the Phyllostomini tribe is confirmed by the use of Carollia brevicauda and Phyllostomus hastatus probes 10,11 .
The phylogenetic relationships of several Myomorpha rodents have been clarified by whole chromosome probes of Mus musculus (MMU; house mouse; Muridae) 12 , however MMU karyotypes are highly rearranged in comparison with Sigmodontinae and few syntenies are observed (e.g., MMU 3/18 and 6/12) 13 .Therefore, the use of whole chromosome probes from a Sigmodontinae taxon gives more accurate information of conserved syntenies and the phylogenetic relationships within this group.This has been observed in the Akodontini tribe (Sigmodontinae, Cricetidae), in which Hylaeamys megacephalus (HME) whole chromosome probes were used to identify chromosomal signatures that reinforce the weakly supported phylogenetic relationships within this tribe 6 .Further, Pereira et al. 14 applied HME whole chromosome probes to Necromys lasiurus and Akodon diauarum (Sigmodontinae), a previously hybridized species using MMU probes 13 , and the results from both probe sets were linked together, allowing MMU syntenic blocks to be identified within Sigmodontinae species that were already hybridized with HME probes, such as MMU5/9, MMU5/7/19, MMU5/10, MMU3/18, MMU8/13 and MMU6/12 14 .In this work, Pereira et al. 14 also identified syntenic blocks shared by representatives in both families (Cricetidae and Muridae), such as MMU5/6, MMU1/17, MMU10/17 and MMU 12/17, and are an indicative of part of the ancestral Eumuroida karyotype (AEK).
Chromosomal traits have been used to construct many phylogenies 11,[15][16][17][18][19][20][21] .This strategy may be applied to explain divergence in Myomorpha rodents in which morphological, molecular and chromosomal traits did not evolve at the same rate as in the above groups 12,23 , resulting in different phylogenetic arrangements.
The genus Cerradomys (Rodentia, Sigmodontinae, Oryzomyini) consists of eight species, which exhibit a wide range of karyotypic diversity, with variation in diploid number (2n) from 46 in C. langguthi to 60 in C. akroai, and autosomal fundamental number (FNa) from 54 in C. marinhus to 76 in C. akroai, with variability in 2n and/ or FNa within some lineages 24 .Recently, the genus Cerradomys (Rodentia, Sigmodontinae) has been investigated www.nature.com/scientificreports/by a multidisciplinary approach (karyotypic, molecular and phylogeographic) 24,25 .In order to understand the chromosomal evolution of the group in the light of their molecular phylogeny, Di-Nizo et al. 24 analysed karyotypic (classic cytogenetics and chromosome painting) and molecular (Cytochrome b-Cytb-and concatenated multi-locus; cyt-b, COI, IRBP and i7FBG)) data from eight species, with 2n 50 to 60 and FNa from 54 to 76 24,26 .These authors identified extensive genomic reshuffling as shown by chromosome painting with Oligoryzomys moojeni (Rodentia, Sigmodontinae) whole chromosome probes 27 , and used Cyt-b topology to shed light on chromosome evolution within this diverse group 24 .Indeed, a phylogeographic approach for the genus Cerradomys suggests that climate changes in the Pleistocene and other biogeographic changes that occurred in South America played an important role in diversification of this group, but chromosomal rearrangements may have facilitated the speciation processes 25 .
In view of the independent evolutionary pathways that chromosomal, morphological and molecular traits of Myomorpha rodents can follow, we set out to investigate if the phylogenetic relationships using chromosomal data corroborated the radiation of Cerradomys taxa recovered by the molecular phylogeny.To achieve this goal, we constructed a chromosomal phylogeny using data from chromosome banding and chromosome painting with HME whole chromosome probes 28 in Cerradomys scotii, C. subflavus and C. vivoi, plus those applied in C. langguthi by Nagamachi et al. 28 .Here, we discuss karyotypic evolution within the genus, the role of chromosomal rearrangements in speciation, and compare our results with the other 21 Sigmodontinae taxa analyzed using the same set of probes 6,14,21,[28][29][30][31][32][33][34] .

Classic cytogenetics
Cerradomys scotii (CSC) has a 2n = 58/FNa = 70 karyotype; the autosomal set consists of 7 meta/submetacentric pairs (1-7) and 21 acrocentric pairs (8-28); the X chromosome is a large submetacentric (Fig. 1a); constitutive heterochromatin (CH) is distributed at the centromeric region of all autosomes; pairs 1, 3 (small submetacentrics) and the X chromosome exhibit large blocks of CH in their short arms (Fig. 2a).Cerradomys subflavus (CSU) has a 2n = 54/FNa = 62 karyotype; the autosomal set consists of 5 meta/submetacentric pairs (1-5) and 21 acrocentric pairs (6-26); the X and Y chromosomes are medium acrocentrics (Fig. 1b); CH is distributed at the centromeric region of all autosomes, except pair 3; the X chromosome exhibits CH at the centromeric region, and the Y chromosome exhibits CH at the distal region of the long arm (Fig. 2b).Cerradomys vivoi (CVI) has a 2n = 50/ FNa = 64 karyotype; the autosomal set consists of 8 meta/submetacentric pairs (1-8) and 16 acrocentric pairs (9-24); the X chromosome is a large acrocentric, and the Y chromosomes is a medium acrocentric (Fig. 1c); the CH is distributed at the centromeric region of all autosomes, except pairs 1-4; the X chromosome exhibits CH at the proximal region, and the Y chromosome exhibits a block of CH at the distal region of the long arm (Fig. 2c).

Regions of homology between MMU and HME
An analysis of 58 rodents hybridized by HME and MMU whole chromosome probes from the Cricetidae (Arvicolinae, Cricetinae, Neotomyinae, Sigmodontinae) and Muridae (Murinae, Deomyinae, Otomyinae) families was conducted (Additional File 2: Table S2).A homology was established between the two sets of probes using the karyotype of Necromys lasiurus (NLA, Sigmodontinae) investigated by HME 14 and by MMU probes 13 .Accordingly, we created an idiogram of the NLA karyotype (Fig. 4) 14 , which allowed us to establish homology between the syntenic blocks of the 37 previously analyzed species and the 21 new species included in this study.
The next clade, composed only of Oryzomyini members recovered the genera Cerradomys as monophyletic (97%), with C. scotii (CSC) as sister to the other taxa, followed by C. langguthi (CLA) (69%), and the most derivate branch with C. subflavus (CSU) and C. vivoi (CVI).The other clade split into the genera Oecomys and Neacomys.The genus Oecomys grouped with high support values in terminal branches, grouping O. catherinae from Rio de Janeiro (OCA-RJ) and O. catherinae from Pará (OCA-PA) with 88% of support, followed by O. auyantepui and a polytomy with 96% that included O. paricola cytotypes A, B and C (OPA-A, OPA-B, OPA-C).The final clade is composed of Neacomys taxa with 72% of support, with the first ramification including a polytomy with N. amoenus (NAM), Neacomys sp.E (NSP-E), and N. elieceri (NEL).The next branch includes N. paracou (NPA),  21 and adapted in Oliveira da Silva et al. 33 .Each HME chromosome is shown with a separate color, except the pairs (9,10), (13,22)    , plus two events of amplification of constitutive heterochromatin in the short arms of the X chromosomes of CLA and CSC (Fig. 6).Only five syntenic blocks were observed without detectable rearrangements, hybridized by HME 11, 18, 23, 24 and 26 probes.We also described an exclusive trait for this genus, the chromosomal association HME 19/7/(9,10).We were unable to compare the chromosome painting data obtained in the present study with those previously published by Di-Nizo et al. 24 , since they did not use the entire set of Oligoryzomys moojeni (OMO) whole chromosome probes and, therefore, there were gaps in the karyotypes of Cerradomys taxa analyzed by them (C.marinhus, C. maracajuensis, C. scotii, C. langguthi, C. vivoi, C. goytaca and C. subflavus).Nevertheless, we corroborate the statement by Di-Nizo et al. 24 that centric fusion/fission, centromere repositioning and pericentric inversions were responsible for the complex scenario that generated the karyotypes of Cerradomys.We did not detect any paracentric inversions and this is explained by the different sets of probes used by Di-Nizo et al. 24 (OMO) and the present work (HME).
In light of the fact that the current karyotypes of HME 28 and OMO 36 are the result of independent rearrangements that occurred during the evolutionary process involving syntenic blocks that are shared between species, chromosome origins are also independent.Thus, probes corresponding to isolated chromosomes reflect this pattern as well.In this regard, the use of either HME or OMO whole chromosome probes as comparative reference sets can detect different arrangements of syntenic blocks within the target species.Accordingly, reciprocal chromosome painting revealed multiple translocations between P. roberti (PRO, 2n = 30) and P. goeldii (PGO, 2n = 25♂), responsible for the diversity between these species, with the conservation of only three chromosomes.In a multidirectional FISH analysis of Proechimys gr.goeldii (PGG, 2n = 16♀17♂), the use of PRO probes allowed to identify 16 fusion/fission events, five translocations, and two inversions, whereas the use of PGO probes allowed to detect 10 fusion/fission events and two inversions 37 .In this way, these data demonstrate the different arrangements of syntenic blocks that can be identified for the same species based on the probe set used.
In order to infer the chromosomal evolution of Cerradomys genus, karyotypic data was plotted on a Cyt-b phylogeny 24,25 and on a concatenated phylogeny (Cyt-b, COI, IRBP and i7FBG) 25 , with C. maracajuensis (2n = 56,58/FNa = 58,60) and C. marinhus (2n = 56/FNa = 54) exhibiting the most conserved karyotypes while the remaining taxa underwent a complex karyotypic evolutionary process with a high rate of rearrangements 24 , Figure 6.Idiograms of the karyotypes of Cerradomys langguthi 28 , C. scotii, C. subflavus and C. vivoi (present study).The karyotypic content of chromosomes involved in rearrangements of each species is separated into columns; the syntenic blocks involved in the rearrangements are arranged horizontally.The box contains an idiogram of HME karyotype previously elaborated 21 and adapted in Oliveira da Silva et al. 33 .Each HME chromosome is shown with a separate color, except the pairs (9,10), (13,22) and ( 16 goytaca (2n = 54/FNa = 62,63,66) 25,26,28,38,39 .In C. maracajuensis, predominates pericentric inversions as well as in C. scotti, with fusion/fission events in the latter one; C. langguthi and C. vivoi exhibited several fusion/fission events, while C. goytaca and C. subflavus exhibited fusion rearrangements and peri-paracentric inversions 24 .Therefore, the proposition that Cerradomys has distinct rates of chromosomal rearrangements 24 is in agreement with our results, as we observed a high rate of chromosomal rearrangements that differentiate the karyotypes of CLA, CSC, CSU and CVI from each other.Similarly, in the speciose genus Nannospalax (Rodentia, Spalacidae), the evolutionary processes could not be fully understood 40 , for karyotype evolution was found to be "complicated and variable" with many parallel courses of speciation in this genus, as fusion events would be the evolutionary pattern for N. leucodon and some representatives of N. xanthodon, while fission events would apply to N. ehrenbergi.
We agree that the block MMU 7/19 (AEK1) is conserved in Muroidea 14,35 , as it is present in all subfamilies investigated here (Table 2, Additional File 2: Table S2).We highlight that solely Oligoryzomys flavescens from the Oryzomyini tribe exhibits the MMU 7/19 (HME 3/25), while being absent in all other 17 Oryzomyini taxa investigated here.
The molecular analysis using cyt-b and concatenated multi-locus (cyt-b, COI, IRBP and i7FBG) based on Bayesian Inference (BI) and Maximum Likelihood (ML) analysis 25 recovered the genus Cerradomys as monophyletic, with C. marinhus + C. maracajuensis as sister to a large clade comprising the six remaining species analyzed; this latter clade splits into two major groups, the first containing C. akroai and C. scotii and the second retaining C. langguthi as sister to the following two subclades: C. goytaca + C. subflavus, and C. vivoi.
Although our chromosomal phylogeny has four (CLA, CSU, CSC and CVI; Fig. 5) out of the eight Cerradomys species recovered in the molecular analysis, our sample are included in two out of the three major clades recovered by Di-Nizo et al. 25 .By comparing both topologies, we observed that the chromosomal phylogeny recovered a similar reconstruction as the molecular phylogeny, with CSC as sister to the other taxa, followed by CLA, and the most derivate branch with CSU and CVI.
In total, C. langguthi underwent 9 fusions, 2 fissions, 5 pericentric inversions, 1 centromere repositioning, and 1 CH amplification; C. vivoi underwent 6 fusions, 1 fission and 2 pericentric inversions; C. subflavus underwent 4 fusions, 1 fission and 3 pericentric inversions; C. scotii underwent 1 fusion, 4 pericentric inversions and 1 CH amplification (Fig. 7).We suggest that the karyotypic diversity is associated with species radiation.In fact, Di-Nizo et al. 25 proposed that historical events that occurred in the Pleistocene played an important role in the diversification of Cerradomys, in which chromosomal rearrangements in isolated populations may have triggered the speciation events.This process could lead to a rupture in gene flow in a secondary contact caused by the expansion of derived populations with new karyotypic forms 44 .
As previously stated, in Myomorpha rodents, morphological, molecular and chromosomal traits of the group do not evolve at the same rate 12,22 , as in Oxymycterus (Sigmodontinae), in which species radiation was not followed by chromosomal diversity, as seven out of the 16 valid species exhibit a conserved karyotype, with the maintenance of 2n with 54 chromosomes and small variation in FNa from 60 to 64 6,26,45 .Another situation is when this mismatch in the rate of evolution of different traits results in cryptic species (two or more distinct species, but with similar morphological traits) 46 , as observed in the genera Akodon and Necromys (Sigmodontinae), Lasiopodomys (Arvicolinae), and Nannospalax (Spalacinae) 40,[47][48][49] .
Here, we observed a case in which these three traits coevolved during the speciation process of Cerradomys.Similar results were detected in Neotropical rodents of the genus Neacomys (Sigmodontinae), in which a gradient of chromosomal and molecular differentiation of four candidate species were reflected in morphological differences that validated these taxa as new species and indicated that speciation was linked to chromosomal variability 21,31,50,51 .Also, a meta-analysis approach compared 41 pairs of rodent sister species and found significant differences in the number of chromosomal traits between sympatric and allopatric species 52 , compatible with a direct role of chromosomal rearrangements in speciation.

Conclusions
We herein report that the Cerradomys genus underwent an extensive karyotypic evolutionary process, with multiple rearrangements that shaped extant karyotypes.Our results show that the chromosomal phylogeny corroborates the phylogenetic relationships proposed by molecular analysis in this genus and indicates that chromosomal rearrangements acted in the speciation process, alongside biogeographic changes in South America that shaped the distribution of extant species.

From the 24
HME whole chromosome probes, five (HME 18, 23, 24, 25, 26) hybridized on whole chromosomes and five (HME 6, 7, 12, 20, 21) hybridized to part of one chromosome each on the three Cerradomys species; the HME 15 hybridized on a whole chromosome in CSC, and part of one chromosome in CSU and CVI; ten

Figure 3 .
Figure 3. FISH results obtained from (a) Cerradomys scotii (CSC), (b) C. subflavus (CSU) and (c) C. vivoi (CVI).An asterisk indicates a centromere.Each box corresponds to a chromosomal pair that is composed of more than one HME homologue.Single or multiples images are addressed to exhibit full coverage with HME probes.HME whole chromosome probes are shown in green (FITC) and red (CY3); counterstaining is blue (DAPI).

Figure 5 .
Figure 5.Most parsimonious tree based on matrix of chromosomal characters in Sigmodontinae taxa.Numbers above branches are Maximum Parsimony (MP) bootstrap values analysed on T.N.T.Only values of 50% and above are shown.A scale representing the distance among species based on accumulated character data is displayed.
Figure 6.Idiograms of the karyotypes of Cerradomys langguthi 28 , C. scotii, C. subflavus and C. vivoi (present study).The karyotypic content of chromosomes involved in rearrangements of each species is separated into columns; the syntenic blocks involved in the rearrangements are arranged horizontally.The box contains an idiogram of HME karyotype previously elaborated21 and adapted in Oliveira da Silva et al.33 .Each HME chromosome is shown with a separate color, except the pairs (9,10),(13,22) and(16,17), which have one color each.(H) indicates large block of constitutive heterochromatin.

Figure 7 .
Figure 7. Direction of chromosomal rearrangements in Cerradomys.Fragment of the chromosomal phylogeny obtained in Fig. 5.Only idiograms of the chromosomes involved in rearrangements of Cerradomys taxa are displayed (Node A) and showing direction of chromosomal rearrangements (Nodes B-G) that shaped extant karyotypes of C. scotii (CSC), C. langguthi (CLA), C. vivoi (CVI) and C. subflavus (CSU).Idiograms are shown based on the HME karyotype (see Fig. 6).Each node indicates the number and type of chromosomal rearrangements that occurred during taxa diversification.The box indicates acronyms for chromosomal rearrangements and its respective legends.

Table 2 .
Syntenic 35ocks shared among representatives of the Muroidea Superfamily were detected by whole chromosome probes of Mus musculus (MMU) and Hylaeamys megacephalus (HME).Regions of homology between MMU and HME are established based on Fig. 4 and regions of homology between MMU and AEK (Ancestral Eumuroida karyotype) are established based on Romanenko al.35.Presence of the syntenic block (+).See Additional File 2: TableS2to access all taxa analyzed.